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Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions*

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Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.

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Author contributions: Study design and direction: JZ, KLH. Omics data acquisition: KK, NL. Data analysis: KK, JAK. Bioassays: MRW, KK. Molecular modeling design: RJW. Modeling: OCG.

*

This work was supported by NIH grants P41GM104603 (JZ, KK, JAK), NIH R01 GM100058, and P41 GM103390 (OCG, RJW) and R01 HL069031 (KLH and MRW). D.F. Smith and R.D. Cummings carried out the CFG glycan array analyses at the National Center for Functional Glycomics (Emory University School of Medicine) funded by the NIH grant P41GM103694. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supplemental material.

1

The abbreviations used are:

    IAV

    Influenza A virus

    CAD

    Collision-activated dissociation

    D+R

    NCRD with two amino acid mutations

    ERManI

    Endoplasmic reticulum class I α-mannosidase

    HA

    Hemagglutinin

    HCD

    Higher-energy collisional dissociation

    HILIC

    Hydrophilic-interaction liquid chromatography

    huNCRD

    Wild-type human neck and carbohydrate recognition domain of SP-D

    LC-MS

    Liquid chromatography-mass spectrometry

    MD

    Molecular Dynamics

    MS/MS

    Tandem mass spectrometry

    MS2

    Tandem mass spectrometry

    Phil-82

    A/Philippines/2/1982 (H3N2)

    Phil-BS

    A/Philippines/2/1982/BS (H3N2) (Bovine serum escape mutant)

    PR-08

    A/Puerto Rico/8/1934 (H1N1)

    PTM

    Post-translational modification

    R343V

    NCRD with single amino acid mutation

    rhSPDII

    Recombinant full length human SP-D

    SP-D

    Surfactant protein-D.